Fabrication Of The SF/PLLA Compound Tissue Engineering Scaffolds By Supercritical Fluids Technology

The fabrication of the porous scaffolds for tissue engineering is one of the keyissues in tissue engineering; it includes the choice of biomaterials and the methods ofscaffold preparation. Silk fibroin/poly-L-lactide (SF/PLLA) have been widely studiedas biomaterials for tissue engineering due to their unique properties including cellaffinity, biological compatibility, good mechanic strength and controllablebiodegradability; supercritical fluids have revealed great potential in design of porousscaffolds. In this study, the porous PLLA and SF/PLLA composite scaffolds werefabricated by supercritical CO2phase separation process using ammoniumbicarbonate (AB) particles as porogen, and the biocompatibility, the release of theloading protein and the material-cell interaction of the scaffolds were alsoinvestigated.Firstly, the PLLA porous scaffolds were prepared by supercritical CO2phaseseparation using AB particles as porogen. The effects of PLLA viscosity, AB size andAB/PLLA ratio on the porosity and compressive strength of the PLLA porousscaffolds were studied in a factorial experiment designed by Minitab. The resultsindicated that, when using PLLA solvent of dichloromethane (DCM), PLLA viscosityof3.5dl/g, AB size of300~600μm and AB/PLLA ratio of20, the resulting PLLAporous scaffolds possessed a high porosity(>95%) and a good compressive strength(>100kPa). At the optimized conditions, the effects of operating parameters(temperature, pressure and time) and different kinds of solvents on the structures ofthe porous scaffolds were studied. The results showed that as the temperatureincreased, the interaction between the polymer and the solvent enhanced, the scaffoldtended to form an inhomogeneous net structure; as the pressure increased, the densityand solvent power of CO2increased, thus increasing the porosity and pore size of thescaffold; as the phase separation time increased, polymer lean phase growth timeincreased, consequently enlarging the pore size of the scaffold. It is noticeable that thestructure obtained was strongly dependent on the solvent used. When PLLA was precipitated from the DCM solution, a micro-pore structure was obtained, and thepore wall was thick and dense. However, the scaffolds obtained from the dioxanesolution presented a nanofibrous network structure, which could not be observed inthe PLLA scaffolds precipitated from DCM solution. When the mixture solution ofdichloromethane/dioxane was used, the scaffolds obtained presented a homogeneousmicro-cell structure; the inner pore wall was rough and consisted of nanoscale cells. Itwas also observed that the size of the large pores formed by the porogen was identicalto that of the AB particles. Without further treatment, the DCM and1,4-dioxaneresidues in the scaffolds were only12ppm and17ppm, respectively; which are muchlower than the limit of the USP467Pharmacopoeia (600ppm and380ppm,respectively). These results indicate that supercritical CO2technology is an effectivemethod for producing a scaffold with little or no organic solvent residue. Aftersupercritical CO2processing, there was no change in the chemical composition of thesamples, these results indicate that the supercritical-CO2-assisted phase inversionprocess is a physical process capable of producing PLLA scaffolds. Despite treatingby the supercritical CO2process, the result of X-ray diffraction (XRD) analysisshowed that the PLLA was still in semi-crystalline state; the result of differentialscanning calorimetry (DSC) analysis also indicated that the supercritical process maynot affect the crystallization of PLLA significantly. The activity of lysozyme loadedin PLLA scaffold was kept above95%.Secondly, the porous SF/PLLA scaffolds were prepared by the same process. Atthe first place, the SF nanoparticles were prepared by solution-enhanced dispersion bysupercritical CO2(SEDS). The effects of the nozzle size, pressure, SF concentration,and flow rates of organic solution and CO2on the properties of SF nanoparticles werestudied. The results indicated that when setting high-pressure vessel at10MPa and35℃, the SF nanoparticles obtained from the0.5%(w/v) SF solution passed throughthe nozzle (inner diameter of150μm) at a flow rate of0.5mL/min presented a goodspherical shape with a mean size of about296.5nm. Zeta potential analysis showedthat the nanoparticle surface possessed powerful negative ions. No change in thechemical composition of the samples occurred during the supercritical process, whichrevealed that it was a physical process to produce SF nanoparticles. XRD and DSC analysis showed that crystalline degree and Tg appeared a minor raising anddecomposition temperature were declined. With the increasing in drug loading, thedrug load increased, while the encapsulation efficiency decreased. The maximumdrug load and encapsulation efficiency of SF nanoparticles were12.7%and74.4%,respectively. Lysozyme-loaded SF nanoparticles possessed a sustained-release effectand the activity of lysozyme was well maintained. Then, the SF/PLLA compositescaffolds were prepared by adding the SF nanoparticles. With the increasing incontent of silk nanoparticles, the porosity of the SF/PLLA composite scaffoldsdecreased from94.5%to91.8%, while the compressive strength increased from123.5kPa to176.6kPa. The sustained-release efficacy of lysozyme from the compositescaffolds was improved than that from SF nanoparticles, and the percentage of drugreleased was54.5%at650hours. The concentration of lysozyme released was in alinear relationship with the drug load.Thirdly, the biocompatibility of SF/PLLA composite scaffolds was evaluated.The cytotoxicity of the composite scaffolds was low with a cell relative growth rate ofabove80%, which belongs to level one. Acute toxicity test showed that no poisoningor death happened within the setting test doses in the mice injected by tail vein. Cellhemolysis test showed the hemolysis ratio of the composite scaffolds was lower than1.5%, which demonstrated no hemolysis effect. Dynamic clotting time test showedthat the scaffolds prolonged the coagulation time, which meets the requirements ofbiomaterials for tissue engineering.Finally, the effects of the SF/PLLA ratio and nanotopography structure of thePLLA scaffolds on cells were investigated. The results indicated that the addition ofSF nanoparticles in the PLLA scaffolds improved the surface roughness and the cellaffinity. With the increasing in content of the SF nanoparticles, the protein adsorption,cell adhesion and cell secretion increased and then decreased, and the influence wassignificantly different compared with the PLLA porous scaffolds (p<0.01). Thenanotopography of the scaffolds has a significant influence on the protein adsorption,cell adhesion and cell secretion (p<0.01). Nanofibrous structure of the PLLAscaffolds possessed a high specific surface, which was in favor of protein adsorptionand L929cell adhesion compared with the smooth surface structure. The macro-pore structure of the PLLA scaffolds favored the protein transmission and cell penetration,there was a significant difference among the scaffolds with different structures(p<0.01).